Photoaffinity labelling has been used for at least 20 years in the identification of specific receptor sites on cell surfaces. Several cell surface receptor sites have been identified by this method; such sites include those for acetylcholine, insulin, glucagon, calmodulin and epidermal growth factors, among others. Heterobifunctional photoaffinity labeling reagents transfer a label to a desired receptor site via the natural ligand for the receptor. The reagent is attached to the ligand in a reaction utilizing one of the two different functional groups of the reagent. Usually, the remaining functional group of the photoaffinity reagent is the photoreactive group which remains inert until activated by light. When the ligand carrying the reagent is introduced to the receptor, a light source is used to activate the photoreactive functional group of the reagent which then covalently binds to the surrounding receptor site. The resulting labelled receptor can then be identified using various well-known detection techniques, depending on the type of label used.
By definition, a heterobifunctional photoaffinity labelling reagent has two different functional groups, one for binding the reagent to a ligand, and the other for binding to the receptor for that ligand. At least the latter functional group is photosensitive. Such a reagent also carriers a detectable label that can be used as a marker for the site where the reagent-bound ligand binds to the ligand's receptor. The reagent should also be cleavable to enable the bound receptor and ligand to be separated, if desired. A cleavable reagent can be separated from the ligand after binding to the receptor. The ligand can then be removed, while the label remains bound to the receptor site.
Two types of photosensitive functional groups are generally available. Azide derivatives and diazo derivatives.
Azide derivatives, such as aryl azido groups, produce nitrenes upon photoactivation. Nitrenes are highly desirable in labelling reactions because they have a half life on the order of 10.sup.-2 to 10.sup.-4 seconds, are highly reactive and non-selective. Diazo derivatives produce reactive carbenes upon photolysis.
Currently available conventional functional groups are used to covalently attach the photoaffinity reagent to amino or sulfhydryl groups of the ligand prior to photoactivation at the receptor site. Disulfide bonds can be provided in the photoaffinity reagent as a functional group to react with available (or added) SH (sulfhydryl or thiol) groups on the ligand. A disulfide exchange reaction covalently binds the photoaffinity reagent to the ligand carrying the SH group. Disulfide bonds make useful functional groups because such a bond can be easily broken under reducing conditions, imparting selective cleavability to the reagent.
It is also desirable to provide a photoaffinity reagent having charged side groups available for solubility of the reagent in aqueous media such as physiological solutions to enable use of the reagent with live cells.
Heterobifunctional photoaffinity labels are very useful in identifying receptor sites, because the labelling is highly specific, and can be achieved directly on intact, live cells under physiological conditions.
Labelling the ligands with radioisotopes is known to facilitate detection and identification of photoaffinity-labelled ligands/receptor complexes. However, some ligands cannot be radiolabelled because they lack suitable reactive groups for attaching the radioactive marker. In addition, some ligands are susceptible to denaturation during the labelling with the radioactive marker and do not bind to their receptor. Incorporating the radioactive marker directly into the heterobifunctional photoaffinity reagent eliminates these problems. See, e.g., Ji and Ji, Analytical Biochemistry, 121, 286-289 (1982) describing the labelling of photoaffinity reagents with reactive radioactive markers such as Na.sup.125 I. However, this technique results in the production of several radioactive reaction products having the marker incorporated at varying points on the reagent, and of varying specific activities which make it difficult to identify the receptor site.
Chong & Hodges (J. of Biological Chemistry, 256 5064-5070 (1981)) synthesized a heterobifunctional photoaffinity reagent utilizing tritiated glycine ([2-.sup.3 H] glycine) as a reactant. The structure of the Chong & Hodges reagent is shown below: ##STR1## Their purpose was to incorporate the radioactive marker during synthesis of the reagent in order to avoid the problem of variable marker incorporation and variable specific activity. However, tritium has a low specific activity [30-60 Ci/mmol] and is counted with less than 50% efficiency by scintillation counting. Therefore, it is difficult to detect in receptor site identification. Thus, while Chong & Hodges solved one problem, they created another.
Synthesis routes for heterobifunctional photoaffinity reagents are usually multistep and produce a low yield of the desired product.
Although reagents exist that have the three features referred to above, e.g. an aryl azido group, a disulfide bond, and charged groups for aqueous solubility (see Chong and Hodges, J. of Biological Chemistry, 256 5064-5070 (1981)), before the present invention there were no photoaffinity reagents that combined these features and additionally incorporated a radioactive marker of high specific activity. Nor were there any such reagents that could be synthesized easily and inexpensively in a single step with high yield.
It is an object of the present invention to provide a radioactive, cleavable, heterobifunctional photoaffinity reagent that can be prepared inexpensively and with high yield in a one-step synthesis from readily available starting materials.
It is another object of the present invention to provide a radiactive, cleavable, heterobifunctional photoaffinity reagent prepared with a radioisotope of high specific activity.
These and other objects of the invention will become apparent to those skilled in the art from the following detailed description of the invention, the accompanying claims and the appended drawings.